Thies, Jennifer et al. (2021) International Worm Meeting "An Environmental Contributor of Neurodegeneration impacts lifespan through disruptions in AMPK Signaling in C. elegans"

Caldwell, Kimberlee, Kim, Hanna, Thies, Jennifer, Caldwell, Guy

[International Worm Meeting, 2021]

Parkinson's Disease (PD) is characterized by the loss of dopaminergic (DA) neurons. Only 5-10% of PD cases have a direct genetic origin; however, exposure to herbicides, pesticides, and interaction with the soil are all potential risk factors. A soil bacterium, Streptomyces venezuelae (S. ven), produces a secondary metabolite that causes age- and dose- dependent DA neurodegeneration in C. elegans. Studies from our lab determined that exposure to the S. ven metabolite causes oxidative stress, mitochondrial fragmentation and enhanced reactive oxygen species (ROS). Upon metabolite exposure, significantly more daf-16 accumulates within nuclei. Recently, we discovered that exposure to the S. ven metabolite is associated with hormetic effects. Specifically, at high concentrations of metabolite (20X), we observe dopaminergic neurodegeneration and enhanced ROS in N2 animals, whereas at low concentrations (5X), the neurons did not display neurodegeneration. We then extended these observations to lifespan studies in N2 animals. Notably, the higher concentration (20X) decreased lifespan in wild-type animals whereas the 5X concentration extended lifespan. daf-16 mutants were then examined with both concentrations of metabolite in lifespan assays. Notably, daf-16 mutants displayed no significant differences between solvent and metabolite at both high and low concentrations, suggesting the hormetic response is daf-16 dependent. Considering daf-16 is the primary transcription factor of several key aging pathways, we investigated the impact of S. ven metabolite on the aging process of C. elegans mutants in the insulin signaling pathway. When both daf-2 and age-1 mutants were exposed to the 5X concentration of S. ven, there was no significance difference between treatments. Yet, when daf-18 mutants were exposed to the same concentration, lifespan extension remained. These data suggest that the insulin signaling pathway is impacted following chronic metabolite exposure. Additionally, when exposed to the 20X concentration of S. ven metabolite, aak-2 mutants displayed no significant difference between solvent and metabolite. However, when aak-2 mutants were exposed to the metabolite at the 5X concentration, mutants displayed a decreased lifespan. Taken together, these data indicate that functional aak-2 might be important for increased lifespan when combating toxicants following chronic exposure.

Willicott, Corey et al. (2021) International Worm Meeting "The mitochondrial unfolded protein stress response is impacted by alpha-synuclein"

Thies, Jennifer, Caldwell, Guy, Willicott, Corey, Caldwell, Kim

[International Worm Meeting, 2021]

PD pathogenesis is associated with misfolding of a-synuclein (a-syn) and mitochondrial dysfunction. Notably, a-syn was shown to interact with, and disrupt, TOMM20, a mitochondrial outer membrane protein. This interaction causes a mitochondrial protein import block, reduced respiration and enhanced generation of reactive oxygen species in cell culture and mammalian brain studies. Our lab extended these findings by reporting that UPRmt stress was increased following overexpression of a-syn variants within the intestine. This was examined via ATFS-1-dependent transcriptional upregulation with the established reporter for the UPRmt, Phsp-6::GFP. These a-syn variants were wild-type human a-syn (WT), two disease variants (A53T and A30P), and a deletion disrupting an N-terminal mitochondrial targeting sequence (delta1-32). The disease variants and the N-terminal mutant induced significantly higher UPRmt activity during both larval development and in adults whereas WT a-syn only induced the UPRmt more than controls in larval development. To determine if UPRmt regulation occurs differently for various familial a-syn variants, the transgenic strains were subjected to RNAi of clpp-1, haf-1, and lonp-1 and then re-analyzed for UPRmt stress. Following knockdown of lonp-1, all the transgenic worm strains displayed significantly enhanced UPRmt activity compared to their EV RNAi controls. Additionally, all variants, except the delta1-32 line displayed enhanced UPRmt stress following knockdown of haf-1. One striking difference in these data, however, was that WT and A53T a-syn-expressing worm lines displayed increases in UPRmt stress between larval and adult stages, while the other two lines did not display increases in UPRmt stress over time. It is interesting to note that the protein pool for at least a subset of WT and A53T a-syn is likely localized to mitochondria, based on previously published work, while A30P and delta1-32 a-syn will remain in the aqueous cytoplasm. These studies were extended to neurons, where knockdown of clpp-1, haf-1, and lonp-1 was performed exclusively in dopaminergic neurons of animals overexpressing WT a-syn. Enhanced neurodegeneration was observed after depletion of clpp-1 and haf-1, whereas knockdown of lonp-1 was similar to EV control. In total, these data suggest that WT and A53T a-syn enter mitochondria where CLPP-1 mitochondrial protease and HAF-1 matrix peptide exporter are involved in their degradation and removal.

Yan, Xiaohui et al. (2011) International Worm Meeting "Identification of genetic modifiers for amyloid-beta toxicity in a C. elegans Alzheimer's disease model."

Yan, Xiaohui, Caldwell, Guy, Knight, Adam, Caldwell, Kim

[International Worm Meeting, 2011]

It is well known that insulin signaling pathways regulating longevity and healthspan are conserved from invertebrates to mammals. In humans, several age-related diseases are associated with protein misfolding or aggregation, including neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Others and we have shown that reduced insulin signaling attenuates the protein misfolding or cytotoxicity associated with poly-glutamine (polyQ) aggregation in HD, amyloid b (Ab) aggregation in AD, and a-synuclein aggregation in PD C. elegans models. Based on the similarity of the effect of insulin signaling on the modulation of misfolded protein toxicity, we tested the hypothesis that a subset of the aging-associated genetic modifiers of PD-related phenotypes identified in our lab will also work for AD. By using a C. elegans AD model in which temperature-inducible muscle expression of human Ab42 transgene leads to a reproducible paralysis phenotype upon temperature upshift, we identified seven candidate genes that accelerate Ab-induced paralysis upon RNAi knockdown. These modifiers include components of ER associated protein degradation (ERAD) and select metabolic factors, including two genes encoding independent but functionally related enzymes, the serine hydroxymethyltransferase (SHMT) and the aminomethyltransferase of the glycine cleavage system (GCS). The reactions catalyzed by SHMT and GCS generate one-carbon units for folate one-carbon metabolism whose normal function supports a variety of cellular activities. This study provides the first evidence of a role for SHMT and GCS in modulating a-synuclein and Ab related cellular toxicity. Interestingly, prior reports have shown that abnormal homocysteine level could affect epigenetic modulation of AD gene activity and that folate deficiency may also influence Ab-induced neurotoxicity. Taken together, these findings expand our mechanistic understanding of the well established, but poorly understood, interface between metabolism and age-associated neurodegenerative diseases.

Gaeta, Anthony et al. (2021) International Worm Meeting "Dietary Composition Modulates Neurodegeneration in a C. elegans Parkinson's Disease Model"

Caldwell, Kim, Caldwell, Guy, Keogh, Candice, McKay, Luke, Gaeta, Anthony

[International Worm Meeting, 2021]

The diet of any organism is crucial in providing the energy necessary to not only exist and persist in the environment, but to overcome stress when challenged. One such type of stress comes in the form of Parkinson's disease (PD), which is characterized by the progressive loss of dopaminergic (DA) neuron function and integrity. Although it has been reported and observed that diet type has wide ranging effects on phenotypes in both humans and animal models, the effect that diet has on the progression of DA neuron loss in PD is largely unknown. However, evidence supporting a connection between the brain and the gut microbiome has become increasingly relevant to neurodegenerative diseases. Furthermore, the effect of parental diet on attributes of PD in subsequent generations has not been adequately investigated. The findings of this study indicate that a non-standard diet for Caenorhabditis elegans (C. elegans) has an impact on PD-associated pathology in this animal, in a transgenerational manner. Specifically, feeding worms an alternative bacterial diet of HB101 E. coli, as opposed to the laboratory standard OP50 E. coli, promotes significant, transgenerational protection of DA neurons in response to the stressor and pathological hallmark of PD, a-synuclein (a-syn) overexpression and accumulation. This protection is shown to be dependent on the normal function of Systemic RNA Interference Defective (SID) proteins, including SID-1, SID-2, and SID-3. These proteins are involved in the ability of double-stranded RNAs (dsRNAs) to traverse cellular boundaries, systemically, and silence target genes in C. elegans to varying degrees. Comparative transcriptomic analysis between worms reared on OP50 vs. HB101 E. coli in an a-syn model background has revealed genes and pathways associated with a wide range of processes. These included genes encoding proteins involved in metabolism, cell signaling, development, transcription, stress responses, and transmembrane transport, all of which were observed to be differentially expressed in worms reared on HB101 E. coli. Subsequent functional analysis of select targets by RNA interference revealed the contribution of individual genes to neuroprotection against a-syn-mediated neurodegeneration. This research lays a foundation for future investigation of the subtle distinctions in diet that can impact conserved pathways modulating neuron survival in response to stress.

Ray, Arpita et al. (2011) International Worm Meeting "Characterizing an uncharacterized protein: F16A11.2, a neuroprotective gene product with a putative role in RNA localization."

DeLeon, Susan M., Caldwell, Kim A., Ray, Arpita, Caldwell, Guy A.

[International Worm Meeting, 2011]

Our studies use C. elegans as an animal model for Parkinson's disease (PD) to identify genetic factors which may impact this condition. PD is characterized by the degeneration of dopaminergic (DA) neurons and the formation of protein inclusions that contain the a-synuclein (a-syn) protein. Overexpression of human a-syn specifically in the eight DA neurons of C. elegans causes neurodegeneration in an age- and dose-dependent manner. The F16A11.2 gene product was found as one of several significant modifiers in an RNAi screen for functional effectors of a-syn toxicity (Hamamichi et al., 2008 PNAS). F16A11.2 is the worm homolog of the human HSPC117 protein (also identified as C22orf28) and shares approximately 75% sequence identity to its human homolog. Interestingly, HSPC117 was determined to be a component of RNA trafficking granules in neurons, along with other proteins, such as CGI-99 (an mRNA transcriptional modulator) and DDX1 (an RNA binding protein). Accordingly, HSPC117 has the ability to bind to AU-rich elements within mRNA. The RtcB domain in F16A11.2 is highly conserved and, based on studies of its bacterial homolog, could represent an uncharacterized RNA modification enzyme. Here, we show that overexpression of the F16A11.2 cDNA in DA neurons not only significantly protected these cells from a-syn neurotoxicity but also that neuronal-specific RNAi knockdown of this target results in enhanced neurodegeneration. Similar results were obtained when worms were exposed to the DA neurotoxin, 6-hydroxydopamine (6-OHDA). Result of yeast two-hybrid (Y2H) screen point to a possible interaction of the F16A11.2 protein with several cytoskeletal factors including actin (ACT-1) and alpha-tubulin (TBA-1). We conducted double knockdown studies between F16A11.2 and those Y2H hits that have been shown to be expressed in neurons and/or have an RNA binding domain, using dopamine neuron-specific RNAi. Our preliminary findings indicate genetic interaction with F59G1.4, a worm homolog of the ceramide glucosyltransferase, DDX-1 and mir-2. As we continue to evaluate other positives from Y2H screening, we are also investigating the possible role of F16A11.2 in RNA trafficking by analyzing P granule localization. Collectively, these studies serve to illuminate the role of this evolutionarily conserved, but previously uncharacterized protein, coincident with the goal of uncovering new pathways associated with neuroprotection.

Griffin, Edward F et al. (2013) International Worm Meeting "Functional analysis of VPS41-mediated protection from b-Amyloid cytotoxicity."

Caldwell, Guy A, Caldwell, Kim A, Griffin, Edward F, Gilmartin, Christopher

[International Worm Meeting, 2013]

According to the Alzheimer's Association 2012 report, 13% of Americans over 65 years of age suffer from Alzheimer's Disease (AD), resulting in an estimated cost of 200 billion dollars for related health care. Additionally, AD is the most prevalent dementia, and the sixth leading cause of death in the United States. Thus, investigating mechanisms of pathophysiology and identifying potential therapeutic targets for AD is significant. AD is characterized by the formation of plaques, composed primarily of b-amyloid 1-42 (Ab) in the brain, resulting in neurodegeneration. Our lab has observed that over-expression of VPS41 in C. elegans provides neuroprotection from Ab toxicity, and that deficiency of VPS41 in worms expressing Ab increases the toxicity of Ab (unpublished data). In yeast, VPS41 has been demonstrated to function in the tethering of vesicles, late endosomes, and AP-3-coated vesicles from the late Golgi, to the lysosome for degradation. Previously, our lab has shown that over-expression of human VPS41 is neuroprotective in a transgenic worm model of Parkinson's Disease, wherein dopaminergic neurodegeneration is induced by a-synuclein overexpression (Harrington et al., 2012, J. Neurosci.). VPS41-mediated neuroprotection from a-synuclein was dependent on the phosphorylation of VPS41 as well as its interactions with AP-3d, RAB7, VPS core proteins, and VPS39. Through this study, and others, we can conclude that VPS41 has a role in lysosomal trafficking. Yet, how this specifically relates to the processing and ameliorates the cellular impact of neurotoxic proteinaceous aggregates is undetermined. Here we report the results of a systematic RNAi screen whereby we knocked down the core components involved in lysosomal trafficking and categorized their requirement for Ab protein toxicity. Preliminary results indicate that Ab is trafficked to through the endosomes rather than through AP-3-coated vesicles from the late Golgi. In this regard, further analysis of functional effectors of Ab protein processing via the lysosomal pathway, along with subsequent evaluation in our worm neuronal model of AD (Treusch et al, 2011, Science), will assist in the elucidation of the underlying mechanism involving VPS-41.

Yan, Xiaohui et al. (2013) International Worm Meeting "Genetic modifiers of amyloid-beta toxicity in C. elegans Alzheimer's disease models."

Knight, Adam L., Caldwell, Kim A., Yan, Xiaohui, Caldwell, Guy A.

[International Worm Meeting, 2013]

The insulin/insulin-like growth factor 1 signaling (IIS) pathway regulates both aging and proteotoxicity and is conserved from invertebrates to mammals. In humans, several age-related neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD) are associated with protein misfolding or aggregation. Others and we have shown that the reduction in IIS ameliorates the proteotoxicity associated with amyloid b (Ab) aggregation in AD, and a-synuclein (a-syn) aggregation in PD C. elegans models. Therefore, we hypothesized that there could be common cellular mechanisms to maintain the proteostasis for counteracting a-syn and Ab misfolding during aging. In this regard, we screened sixty age-related genetic factors that modify a-syn aggregation, in a C. elegans AD model in which temperature-inducible muscle expression of human Ab42 leads to a reproducible paralysis phenotype. We identified seven genes that accelerate Ab-induced paralysis upon RNAi. These modifiers include components of the protein clearance and select metabolic factors, including two genes encoding independent but functionally related enzymes, the serine hydroxymethyltransferase (SHMT) and the aminomethyltransferase (AMT) of the glycine cleavage system (GCS). The reactions catalyzed by SHMT and GCS generate one-carbon units for folate-dependent one-carbon metabolism whose normal function supports a variety of cellular activities. When AMT was overexpressed in our C. elegans neuronal AD model, where Ab42 accumulation induces an age-dependent degeneration of glutamatergic neurons (Treusch et al., 2011, Science), the neuronal loss was significantly rescued. This study provides the first evidence of a role for SHMT and GCS in modulating a-syn and Ab related proteotoxicity. Interestingly, alterations in one-carbon metabolism have been linked to epidemiological and genetic association studies for AD risk, yet, the underlying cellular mechanisms are largely unknown. Taken together, these findings will potentially expand our mechanistic understanding of the well established, but poorly understood, interaction between metabolism, especially the folate-dependent one-carbon metabolism, and age-associated neurodegenerative diseases.

Berkowitz, Laura A. et al. (2009) International Worm Meeting "Dopamine Signal Transduction Components Alter Neurodegeneration in a Parkinson's Disease Model."

Hamamichi, Shu, Caldwell, Kim A., Berkowitz, Laura A., Caldwell, Guy A.

[International Worm Meeting, 2009]

Improvements to the diagnosis and treatment of Parkinson''s disease (PD) are contingent upon knowledge about susceptibility factors that render populations at risk. Our work has focused on a functional analysis that exploits the experimental attributes of C. elegans to accelerate the translational path toward identification of effectors of dopamine (DA) neuron degeneration in vivo. C. elegans has only 8 DA neurons, providing a simple system in which to study PD-like neurodegeneration with unparalleled accuracy in quantitation of neuron survival in statistically substantial numbers of test animals. Our prior application of C. elegans in this directed manner has been successful in rapidly predicting genes that have significant consequences for neuronal survival in mammalian systems (Gitler et al., 2008; PNAS; Gitler et al., 2009, Nat. Genetics) Human a-synuclein, a protein found in the PD-associated aggregates called Lewy bodies, when overexpressed in C. elegans DA neurons, causes them to degenerate in an age- and dose-dependent manner. GAIP Interacting Protein C-terminus (GIPC) was identified in a large-scale RNAi screen for genes that alter both a-synuclein aggregation and DA neuron degeneration (Hamamichi et al., 2008, PNAS). GIPC interacts with the RGS-GAP (GTPase Activating Protein) GAIP, which attenuates G-protein signals. Additionally, GIPC interacts with a number of G-coupled receptors including the dopamine receptor, where it has been proposed to play multiple roles in DA signal transduction such as: 1) membrane recruitment of GAIP (which down-regulates DA signaling); 2) DA receptor internalization; and 3) DA receptor recycling. Here we report that genetic loss of GIPC enhances neurodegeneration, whereas overexpression (OEx) is neuroprotective. To determine what GIPC-dependent function(s) and possible interactor(s) is involved in its neuroprotective capacity, proteins that act in the above pathways are being analyzed via genetic knockouts and DA neuron-specific overexpression. This analysis has revealed that loss of presynaptic dopamine D2 receptors (dop-2) increases degeneration. Moreover, the neuroprotective capacity of GIPC OEx is eliminated in a dop-2 mutant background, suggesting that GIPC protects through the DA receptor, as opposed to acting via any number of other potential receptors. Loss of the C. elegans homolog of GAIP, rgs-2, is also protective while overexpression of this GIPC interactor enhances DA neuron loss. Taken together, these results suggest that components of the DA signaling cascade contribute to neuron survival. Further analysis of GIPC activity may provide mechanistic insights into PD.

Tucci, Michelle L. et al. (2011) International Worm Meeting "Investigation of the neuroprotective role of GIPC in a C. elegans model of Parkinson's Disease."

Berkowitz, Laura A., Caldwell, Kim A., Caldwell, Guy A., Tucci, Michelle L.

[International Worm Meeting, 2011]

Our work has focused on functional analyses designed to identify effectors of dopamine (DA) neurodegeneration using the model organism Caenorhabditis elegans (C. elegans). When human alpha-synuclein (a-syn), a protein found in Parkinson's Disease (PD)-associated aggregates, is overexpressed in the C. elegans DA neurons, age and dose-dependent neurodegeneration takes place. In a large-scale hypothesis-based RNAi screen for gene products that enhance a-syn aggregation and DA neuron degeneration, GAIP Interacting Protein C-terminus (GIPC) was identified as a neuroprotective factor (Hamamichi et al., 2008, PNAS). Evidence reveals a role for GIPC in transduction through recruitment of the G protein signal attenuating GAIP protein. The general association of GIPC with many receptors and transporters localized at the plasma membrane that undergo internalization, recycling and/or degradation provide a large spectrum to identify which of these functional roles GIPC may play in protecting the DA neurons from a-syn induced toxicity. We report that both the genetic loss and overexpression of C35D10.2, the C. elegans homolog of GIPC, is neuroprotective. Use of genetic knockouts, DA neuron-specific RNAi methods and DA neuron overexpression of candidate genes involved in the DA signaling cascade, endocytic trafficking and recycling/degradation allows us to determine GIPC-dependent functions and interactors involved are in neuroprotection. Preliminary analysis shows that loss of presynaptic D2 autoreceptor (DOP-2) has no effect on a-syn induced degeneration, nor does it alter the level of protection with the genetic loss of GIPC. However, the DA signal is required for protection with the GIPC overexpression. Additional neurotoxin (6-OHDA) and reporter assays suggest possible down regulation of the dopamine transporter protein, DAT-1, that may, in turn, influence cytosolic DA levels. These results indicate that components of DA neurotransmission, as well as the endocytic pathway may contribute to neuronal survival. Taken together, these studies further provide mechanistic insights into factors impacting DA neuron survival and Parkinson's Disease.

Willicott, Karolina et al. (2021) International Worm Meeting "A Genetic Screen for Identification of UPRmt Effectors Associated with a-synuclein Neuroprotection in C. elegans"

Davidson, Rose, Caldwell, Kim, Greene, Madeline, Willicott, Karolina, Caldwell, Guy, Bolden, Rachel

[International Worm Meeting, 2021]

Parkinson's disease (PD) is characterized by neurodegeneration of the central nervous system, leading to impaired motor skills and bodily functions. Here, we look at two components of PD pathology: One is the protein alpha-synuclein (a-syn) that is found within Lewy Bodies and is often prone to misfolding and aggregation. The other is mitochondrial dysfunction via a dysregulated Unfolded Protein Response (UPRmt). Under non-stress conditions, the Activating Transcription Factor Associated with Stress (ATFS-1) is imported into the mitochondria and degraded. During acute stress, mitochondria employ the (UPRmt), that, with the help of ATFS-1, coordinates nuclear expression of chaperones and proteases, that translocate to the mitochondria and remove damaged or unfolded proteins. However, during a long-term genetic stressor such as a-syn, this system becomes dysregulated. We hypothesize that a physical blockade is created when a-syn interacts with the outer mitochondrial membrane protein, TOMM-20, and that this blocks mitochondrial import, leading to proteostatic imbalance. Our research shows that the loss-of-function mutant of atfs-1 is highly neuroprotective against a-syn-induced neurodegeneration. Therefore, we used atfs-1(lof); a-syn animals in an F3 EMS mutagenesis screen to identify UPRmt effectors associated with a-syn neuroprotection. We have screened through ~3,300 genomes. Populations of animals were examined for changes in neurodegeneration levels using a low power, fluorescent, stereomicroscope and then confirmed for neurodegenerative changes with compound microscopy analysis. In the afts-1 (lof); a-syn background, more than half of the animals display neuroprotection in the background and we selected mutants where the dopaminergic neurodegeneration level went back to levels similar to a-syn only levels. Through this screen we initially isolated ~70 mutants. These positive candidates were collected, propagated, and screened over multiple generations for consistent neurodegenerative phenotypes, which ultimately provided us with 24 mutants. Of these mutants, at least 11 independent mutations are expected. After outcrossing, DNA was extracted from the mutants and sequenced using the Illumina platform. SNP analysis is currently being performed using GATK. Mutants obtained from this screen may reveal an uncharacterized compensatory mechanism for UPRmt induction.